Nucleic Acids Research, Vol. 18, No. 21 6445

Cloning,

sequence

and expression of rat cathepsin D

Nigel P.Birch* and Y.Peng Loh Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Submitted September 21, 1990

EMBL accession

Cathepsin D is a member of the family of aspartic proteases and is thought to play a role in the lysosomal-mediated degradation of proteins (1). We have isolated the cDNA for rat cathepsin D from a pituitary cDNA library, screened using oligonucleotide probes synthesized to the conserved active site regions of aspartic proteases (Figure 1). The cDNA contains an open reading frame which encodes for a polypeptide of 407 amino acids. The predicted amino acid sequence shows 83% and 91 % amino acid identity with human (2) and mouse (Birch, unpublished data, 3) CCTATAAGCC GGCGACCTCT GGCATTAAGC TTTGCTCTCT TCGGGCCGTC GCGACC

no.

X54467

cathepsin D. The sequence predicts two N-linked glycosylation sites at amino acid residues 134 and 258. The cDNA (1940 bp HindII-X7hoI fragment) was subcloned into pGem3Z (Promega) and capped RNA prepared from the SP6 polymerase promoter. Incubation in a rabbit reticulocyte cell-free translation system (Promega) demonstrated that the cDNA encoded a -38000 dalton protein, which upon glycosylation increased to 43500 daltons. -

ATG CAG ACC CCC GGC GTC TTG CTG CTC ATT CTC GGC CTC CTG Mot Gln Thr Pro Gly Val Lou Lou Lou I1e Lou Gly Lou Lou

98

GAT GCG TCC TCC TCC GCG CTT ATC AGA ATC CCC CTG CGC AAG TTC ACA TCC ATC CGT CGG ACT ATG ACG GAA GTG GGA GGC TCT GTG GAA Asp Ala Ser Sor Ser Ala Lou Ile Arg I1e Pro Lou Arg Lys Pho Thr Ser Ile Arg Arg Thr Met Thr Glu Val Gly Gly Ser Val Glu

188

GAC CTG ATC CTT AAA GGT CCC ATA ACC AAG TAC TCC ATG CAG TCA TCT CCT AGG ACC AAG GAG CCA GTG TCA GAG TTA CTA AAA AAC TAC Asp Leu Ile Lou Lys Gly Pro Ile Thr Lys Tyr Ser st Gln Sor Sor Pro Arg Thr Lys Glu Pro Val Sor Glu Lou Lou Lys Asn Tyr

278

CTG GAT GCC CAG TAC TAT GGT GAG ATC GGC ATT GGG ACT CCC CCA CAG TGT TTC ACA GTC GTC TTT GAC ACT GGC TCC TCT AAC CTG TGG Lou Asp Ala Gln Tyr Tyr Gly Glu Ile Gly Ile Gly Thr Pro Pro Gln Cys Phe Thr Val Val Phe Asp Thr Gly Ser Ser Asn Lou Trp

368

GTC CCC TCC ATT CAT TGC AAG CTG CTG GAC ATA GCC TGC TGG GTC CAC CAC AAG TAC AAC AGT GAC AAG TCC AGC ACC TAT GTG AAG AAT Sor I1- His Cys Lys Lou Lou Asp I1e Ala Cys Trp Val His His Lys Tyr Asn Ser Asp Lys Bar Ser Thr Tyr Val Lys Asn

458

GGC ACA TCC TTC GAC ATC CAC TAC GGC TCA GGT AGC CTC TCT GGG TAC CTG AGC CAG GAC ACT GTG TCG GTT CCA TGT AAG TCA GAC TTA

548

GGA GGT ATC AAG GTG GAG AAA CAG ATC TTT GGG GAA GCC ACC AAG CAG CCT GGA GTC GTA TTC ATC GCA GCC AAG mTT GAT GGC ATC TTG Gly Gly Ile Lys Val Glu Lys Gln I1e Phe Gly Glu Ala Thr Lys Gln Pro Gly Val Val Phe I1e Ala Ala Lys Phe Asp Gly I1e Lou

638

GGC ATG GGC TAC CCT TTT ATC TCT GTT AAC AAG GTG CTC CCG GTC TTC GAC AAC CTG ATG AAA CAG AAG CTG GTG GAA AAG AAC ATC TTC M1t Gly Tyr Pro Phe I1e Sor Val Asn Lys Val Lou Pro Val Pho Asp AMn Lou Met Lys Gln Lys Lou Val Glu Lys AMn I1e Pho

728

TCC TTC TAC CTG AAC AGG GAC CCA ACC GGG CM CCT GGA GGA GAA CTA ATG CTT GGC GGC ACT GAC TCC AGA TAC TAC CAC GGG GAG CTG Ser Phe Tyr Lou Asn Arg Asp Pro Thr Gly Gln Pro Gly Gly Glu Lou Met Lou Gly Gly Thr Asp S*r Arg Tyr Tyr His Gly Glu Lou

818

TCC TAC CTG AAC GTC ACC CGA AAG GCC TAC TGG CAG GTG CAC ATG GAC CAG CTG GAG GTG GGC AGC GAG CTG ACC CTG TGC AAG GGA GGC Ser Tyr Lou Asn Val Thr Arg Lys Ala Tyr Trp Gln Val His Mtt Asp Gln Lou Glu Val Gly SBr Glu Lou Thr Lou Cys Lys Gly Gly

908

TGT GAG GCT ATT GTG GAC ACA GGG ACG TCT CTT CTG GTG GGG CCT GTG GAC GAG GTG AAG GAA CTA CAG AAG GCC ATT GGG GCA GTG CCT

998

Val Pro

Gly Thr Ser Pbe Asp I1e His Tyr Gly Ser Gly Ser Lou Sor Gly Tyr Lou Bar Gln Asp Thr Val Sor Val Pro Cys Lys SBr Asp Lou

Gly

Cys Glu Ala Ile Val Asp Thr Gly Thr

Sor

Lou Lou Val Gly Pro Val Asp Glu Val Lys Glu Lou Gln Lys Ala 110 Gly Ala Val Pro

GGC GAG TAT ATG ATC CCT TOT GAG AAG GTG TCC AMC CTG CCC ATT ATC ACC TmT AA CTA GOA GC CGA AAC TAT GAA CTIA 10s Gly Glu Tyr Met Il Pro Cys Glu Lys Val Ser Sor Lou Pro Il Il Thr Pho Lys Lou Gly Gly Gln AMn Tyr Glu Lou CCA GAG AMG TAC ATA CTC AAG GTA TCG CAG GCT GGA AAG ACG ATC TGC CTG AGT GGC TTC ATG GGG ATG GAC ATA CCC CCT CCC AGT 1178 Pro Glu Lys Tyr Ile Lou Lys Val Ser Gln Ala Gly Lys Thr Il Cys Lou Ser Gly Phb Met Gly Mst Asp Ile Pro Pro Pro Scr CCG CTC TGG ATC CTG GGC GAT GTC mTT ATT GGC TGC TAC TAC ACC GTG TTT GAC AGA GAa TAC AAT AMG GTC GGC TmT GCC AAG GCT 1268 Pro Lou Trp Ile Lou Gly Asp Val Phe Il Gly Cys Tyr Tyr Thr Val Phb Asp Arq Glu Tyr Asn Ara Vol Gly Phe Ala Lys Ala

CTC ATC CAG Lou Ile Gln

CAC

His GGG

Gly

GCC ACA CTC TAA CTTGC TCCTTCTTCA CTGTCAGGGA ACTGGATCAG AGTCCAGTAG AGAAAGCCAG CCAGCCCCAT CCCTCCACCT GCCCCACTCA CACATATTCA 1375 Ala Thr Lou ***

,CACTCGCCTA GTGTTGCTGG -GCCCTTGGGA GGCCTGGCTG GAGCTGGTCC AGCTGTTCTG TTCTGTGGTC CCTGTCCCTG GGTTCAGATT GCTGCCCTCC GCCTGTCTGA 1485 AGGAGGCCAA GGCCTACCCA

GTACAAMG CTGCCTTTAA AGGCCCCTAC TGGCTTGGTA GCTGCCGGGA TGAATTGTCT TGTCTGCTG CCCTTTGCTG CGTGGGCAGC 1595

ACTCTGAAGC AGGCAAATGG GTCTTGGGAT CCCTCCCAGA AACCTGCTCT GAccAMAccc TCAGGCAGCC TGGGGATGC ACCAAGCTCT ACTGCCCCAC TATCTCTGCC 1705

CTGGCAAAGG CTGAAGGTGA GCAGAAGGAG CAAGAGGACA GGCAAAAC TATATGAMCC TGGGGGGGGT TACCTGGGAC CTGACCCCAC CCTCCTGGGA AGGCATGCTT 1815 CAGCCTGGGG CAGAGGTAGG ATGGCTGACT GGTTTTGGCT GGCCTCTGCT GCCCTCATCC TGGGCTAGTC AGATGGGAGC CAAAGTTGAT ATACAMATAA AGTTGTTTTG 1925

GGCCTCTTTA AAAMMAM AAMAAAA AAAAAAAAAA AAAAMM AAAA

A A

Figure 1. Nucleotide sequence and deduced amino acid sequence of rat cathepsin D cDNA. * To whom correspondence should be addressed

1986

6446 Nucleic Acids Research, Vol. 18, No. 21

Figure 2. Cell-free translation of rat cathepsin D mRNA in a reticulocyte lysate system. Reactions were carried out as suggested by the supplier (Promega) in the presence of [35S]methionine (1000 Ci/mmol; NEN) and processed for SDS polyacrylamide gel electrophoresis followed by autoradiography. Lane A, no exogenous mRNA; Lane B, 1 ;ig rat cathepsin D mRNA; Lane C, 1 jig rat cathepsin D mRNA plus dog pancreas microsomal membranes (Promega). The migration positions of prestained protein molecular weight standards (BRL) are also shown.

ACKNOWLEDGEMENTS We would like to thank Dr Michael Brownstein for the rat pituitary cDNA library and for his continued advice and encouragement.

REFERENCES 1. Tang,J. and Wong,R.N.S. (1987) J. Cell. Biochem. 33, 53-63. 2. Faust,P.L., Kornfeld,S. and Chirgwin,J.M. (1985) Proc. Natl. Acad. Sci. USA 82, 4910-4914. 3. Grusby,M.J., Mitchell,S.C. and Glimcher,L.H. (1990) Nucd. Acids Res. 18, 4008.

Cloning, sequence and expression of rat cathepsin D.

Nucleic Acids Research, Vol. 18, No. 21 6445 Cloning, sequence and expression of rat cathepsin D Nigel P.Birch* and Y.Peng Loh Section on Cellular...
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